The primary transcripts of most eukaryotic structural genes (pre-mRNAs) contain intervening sequences that are removed by RNA splicing. The biochemical mechanisms involved in pre-mRNA splicing and in particular how these mechanisms allow accurate splice-site selection to be achieved are not fully understood. During the past several years we have developed an efficient and versatile system for studying pre-mRNA splicing in vitro. This approach has revealed new information about the biochemistry of pre-mRNA splicing and has allowed for the identification and characterization of probable splicing intermediates. Based upon these results, we have proposed a pathway for splicing of pre-mRNAs. In this proposal we outline experiments that continue and extend these studies. Our objectives are to determine the role of specific cis-acting pre-mRNA sequences in splicing, identify and characterize the biochemical factors that are involved in pre-mRNA splicing, and study the interaction of these factors with the RNA substrate. Finally, we will study in vitro the processing of adenovirus primary transcripts that undergo regulated differential splicing in vivo. By using extracts prepared from viral-infected cells, we hope to duplicate these alternative splicing events in vitro. Such a regulated in vitro processing system should provide a useful model for analyzing the biochemical mechanisms involved in the control of gene expression by differential RNA processing.